Injectable Hyaluronan Hydrogels with Peptide-Binding Dendrimers Modulate the Controlled Release of BMP-2 and TGF-ß1.

BMP-2 and TGF-ß1 released from injectable thermoresponsive hydrogels are studied in the presence and absence of branched macromolecules bearing BMP-2 or TGF-ß1 affinity binding peptides. The synthesized branched macromolecules and the gelling compositions before and after loading with either BMP-2 or TGF-ß1 are characterized physico-chemically and show a significantly lower amount of proteins released in the presence of the affinity binding peptide macromolecules. This study illustrates the potential of affinity binding peptide functionalized dendrimers to modulate the local delivery and availability of growth factors important for musculoskeletal regeneration therapies.

Copper catalyst efficiency for the CuAAC synthesis of a poly(N-isopropylacrylamide) conjugated hyaluronan.

BACKGROUND: Poly(N-isopropylacrylamide) conjugated hyaluronan (HA-pN), a brush-like copolymer system which serves as a polymer vehicle for cellular and drug delivery, has been previously synthesized via the copper catalyzed azide-alkyne reaction (CuAAC) using a combination of copper sulfate and ascorbic acid (CuAsc) as the catalytic system of choice. Bromotris(triphenylphosphine) copper(I) (CuBr(PPh3)3) is an alternative catalytic compound containing a phosphorous ligand which stabilizes copper in the +1 oxidative state in aqueous solvents and can be employed at true catalyst concentrations. OBJECTIVE: CuAsc and CuBr(PPh3)3 were compared for their efficiency; 1) in the synthesis of HA-pN via CuAAC; 2) in producing thermoresponsive compositions and 3) in being extracted from the polymeric compositions. METHODS: The synthesis of the brush copolymer was carried out under strict Schlenk conditions, then characterized by ATR-FTIR, 1H NMR, ICP-SFMS, and rheological measurements. RESULTS: CuBr(PPh3)3 catalyzed CuAAC leads to better grafting in water, at a true catalyst concentration, compared to CuAsc. Polymeric solutions exhibited similar traits of increasing mechanical stiffness with rising temperature. Despite purification via chelation and/or dialysis, residual copper was present in similar concentrations in the final polymers. CONCLUSIONS: In the CuAAC driven copolymer synthesis of the HA-pN, CuBr(PPh3)3 is a better catalyst than CuAsc.

Multivalent dendrimers presenting spatially controlled clusters of binding epitopes in thermoresponsive hyaluronan hydrogels.

The controlled presentation of biofunctionality is of key importance for hydrogel applications in cell-based regenerative medicine. Here, a versatile approach was demonstrated to present clustered binding epitopes in an injectable, thermoresponsive hydrogel. Well-defined multivalent dendrimers bearing four integrin binding sequences and an azido moiety were covalently grafted to propargylamine-derived hyaluronic acid (Hyal-pa) using copper-catalyzed alkyne-azide cycloaddition (CuAAC), and then combined with pN-modified hyaluronan (Hyal-pN). The dendrimers were prepared by synthesizing a bifunctional diethylenetriamine pentaacetic acid core with azido and NHBoc oligo(ethylene glycol) aminoethyl branches, then further conjugated with solid-phase synthesized RGDS and DGRS peptides. Azido terminated pN was synthesized by reversible addition-fragmentation chain transfer polymerization and reacted to Hyal-pa via CuAAC. Nuclear magnetic resonance (NMR), high performance liquid chromatography, size exclusion chromatography and mass spectroscopy proved that the dendrimers had well-defined size and were disubstituted. NMR and atomic absorption analysis confirmed the hyaluronan was affixed with dendrimers or pN. Rheological measurements demonstrated that dendrimers do not influence the elastic or viscous moduli of thermoresponsive hyaluronan compositions at a relevant biological concentration. Finally, human mesenchymal stromal cells were encapsulated in the biomaterial and cultured for 21days, demonstrating the faculty of this dendrimer-modified hydrogel as a molecular toolbox for tailoring the biofunctionality of thermoresponsive hyaluronan carriers for biomedical applications.